Date

Author

Metadata

Abstract

Tuberculosis (TB) remains a major global health issue, especially with the emergence of drug-resistant strains of Mycobacterium tuberculosis , which in some cases are untreatable. Despite many efforts, the need for new drugs and strategies to combat this disease is still clear. The biosynthesis of the three branched-chain amino acids (BCAAs) is essential for the growth of M. tuberculosis. The only enzyme shared in the biosynthesis of all three BCAAs is the ilvE-encoded aminotransferase, IlvE (MtI1vE). MtIlvE is a pyridoxal 5'-phosphate (PLP)-dependent enzyme that catalyzes the final transamination step in the pathway leading to the synthesis of L-isoleucine, L-valine and L-leucine. This thesis focuses on the biochemical characterization of MtI1vE and validation of this enzyme as a target in M. tuberculosis.;MtIlvE catalyzes a ping-pong reaction where the alpha-amino group of L-glutamate is transferred to the PLP cofactor, and then transferred to the cognate alpha-keto acid of the BCAA. Primary and solvent kinetic isotope effects (KIEs) data were determined for the ping reaction and solvent KIEs determined for the pong half-reaction. A 1,3-prototropic shift mechanism was proposed for this enzyme based on the synchronous deprotonation of the C2-H of L-glutamate and protonation of C4' of the PLP cofactor in the ping half-reaction.;MtIlvE is subject to mechanism-based inhibition or "suicide" substrate inactivation. MtIlvE's potential as a drug target has already been demonstrated by its inhibition with aminooxy compounds. D-cycloserine is a second-line TB drug used to treat multidrug-resistant strains of M. tuberculosis (MDR-TB). The drug acts by inhibiting D-alanine D-alanine ligase and the PLPdependent enzyme alanine racemase (AR). MtIlvE is completely inactivated in the presence of both D- and L-cycloserine in a time- and concentration-dependent fashion. L-cycloserine is 40-fold better inhibitor of MtIlvE than D-cycloserine. The minimum inhibitory concentration (MIC) of L-cycloserine is 10-fold lower than D-cycloserine. The 1.7 A crystal structure of the MtIlvE-PMP-D-cycloserine inhibited complex revealed a planar, aromatic cycloserine ring. Mass spectrometry of the PMP-D-cycloserine and PMP-L-cycloserine adducts resulted in the exact same mass suggesting the formation of an isoxazole product. Spectroscopic kinetic data on the formation of the isoxazole products revealed differences between the two stereoisomers. An aromatization mechanism for the inactivation of MtIlvE by D- and L-cycloserines was proposed.;beta-Chloroalanine is one of the many substrate analogues designed to inhibit AR. Recent studies have showed that this compound is a poor inhibitor of M. tuberculosis AR, but potently inhibits the M. tuberculosis L-glutamate racemase. Here, we investigated the ability of beta-chloroalanine enantiomers to inhibit MtIlvE. The stereoisomers of beta-chloroalanine are slow, tight-binding, irreversible inhibitors of MtIlvE, with the L-isomer being a more potent inhibitor of the enzyme. The reaction proceeds via chloride elimination and formation of an alpha-aminoacrylate intermediate, leading to a covalent, irreversibly inhibited complex. Both beta-chloroalanine isomers inhibit the growth of M. tuberculosis, however the L-isomer is 5 times more potent in inhibiting the H37Rv auxotrophic mc23206 strain (DeltapanCD). The supplementation of the growth media with a single BCAA or all three BCAAs rescued the culture growth, suggesting that glutamate racemase may not be the only target of beta-chloroalanine in M. tuberculosis. A chemical mechanism of inhibition and catalysis of MtIlvE by beta-chloroalanine isomers is proposed. In summary, the essentiality, and possibility for small molecule disruption, of the biosynthesis of BCAAs should be explored as a new target for drug development against drug-resistant tuberculosis.